C. elegansis a member of a group of nematodes called rhabditids,
which encompasses a large number of ecologically and genetically diverse species. A new,
preliminary phylogenetic analysis is presented for concatenated sequences of three
nuclear genes for 48 rhabditid and diplogastrid species (including 10Caenorhabditisspecies), as well as four species representing
the outgroup. Although many relationships are well-resolved, more data are still needed
to resolve some key relationships, particularly near the base of the rhabditid tree.
There is high confidence for two major clades: (1) a clade comprisingMesorhabditisParasitorhabditis,Pelodera,Teratorhabditisplus a few other
species; (2) a large clade (Eurhabditis) comprising most of the remaining rhabditid
genera, includingCaenorhabditisand its sistergroupProtorhabditis-Prodontorhabditis-Diploscapter.Eurhabditis also contains the parasitic
strongylids, the entomopathogenicHeterorhabditis,and the
monophyletic groupOscheiuswhich includes the satellite model organismO. tipulae.The
relationships withinCaenorhabditisare well resolved. The
analysis also suggests that rhabditids include diplogastrids, to which the second
satellite model organismPristionchus pacificusbelongs. Genetic
disparity withinCaenorhabditisis as great as that across
vertebrates, suggestingCaenorhabditislineages are quickly evolving, ancient, or both. The
phylogenetic tree can be used to reconstruct evolutionary events within rhabditids. For
instance, the reproductive mode changed multiple times from gonochorism to
hermaphroditism, but only once from hermaphroditism to gonochorism. Complete retraction
of the male tail tip, leading to a blunt, peloderan tail, evolved
at least once. Reversions to unretracted tail tips occurred within both major rhabditid
groups. The phylogeny also provides a guide to species which would be good candidates
for future genome projects and comparative studies.

Caenorhabditis belongs to a large group of primarily
bacteriophagous nematodes called rhabditids (Figure 1). This taxon
comprises a great diversity of species, including some parasites (see A quick tour of nematode diversity and the backbone of nematode phylogeny). Resolving rhabditid phylogeny is key to uncovering how
evolutionary changes occurred in this diversification. A phylogeny for rhabditids also
provides an important foundation for comparative biology using C.
elegans, "satellite" model systems Pristionchus
pacificus, Oscheius tipulae, and other
Caenorhabditis species with sequenced genomes (C.
briggsae, C. remanei, C.
japonica, and Caenorhabditis n. sp. represented by
strain CB5161 among others; see Evolution of development in nematodes related to C. elegans). The selection of appropriate representative taxa for further genome
sequencing projects also depends on an accurate phylogeny.

Phylogenetic analyses using DNA sequences of small subunit ribosomal RNA (SSU rRNA)
genes have revealed some relationships that are inconsistent, as well as some that are
consistent, with earlier phylogenies (Fitch et al., 1995; Blaxter et al., 1998; Sudhaus and Fitch, 2001). Many relationships, however,
have not been sufficiently resolved. It is therefore important to expand the data pool
with regard to both loci and taxa.

Here we report conclusions of an analysis (which will be published elsewhere) using three genes for rhabditids and
their closest relatives. In this phylogeny, several branches are well supported. For
example, most species belong to either of two major monophyletic groups, here called
"Eurhabditis" and "Pleiorhabditis", both of which show high support in this analysis.
Eurhabditis includes strongylids and Heterorhabditis, as well as
Caenorhabditis and its sister taxon, the Protorhabditis group.
There is also some evidence that diplogastrids (e.g., Pristionchus)
are part of rhabditids. As we accumulate more data, we are gaining a clearer picture of
rhabditid phylogeny. There is still much that remains mysterious, however. In
particular, it will be important to establish who are the closest relatives of
rhabditids, to sort out the earliest lineages of rhabditids, and to further elucidate
the relationships among members of the Eurhabditis group. Only after this is achieved
should the much-needed taxonomic revision be initiated. At the moment, however, it is
still too early to attempt such a classification because a number of relationships are
not sufficiently resolved.

A well-resolved phylogeny is an essential step in inferring the evolutionary changes
that have occurred and for discovering possible correlations between such changes. We
provide examples of how the phylogeny so far has provided insight into the evolution of
reproductive mode, morphological evolution in the male tail tip, the evolution of
introns, and molecular divergence. Database and strain resources for rhabditids are
being developed to support comparative work with rhabditids.

2. Rhabditid phylogeny

2.1. Overview of rhabditid relationships

A preliminary phylogenetic hypothesis for representative rhabditid species and
four species from the outgroup is shown in Figure 1. The analysis
is based on DNA sequences from nearly complete small and large subunit (SSU and LSU)
ribosomal RNA (rRNA) genes and a portion of the RNA Pol II (RNAP2) gene (KK and DF,
unpublished; original data and details of the analysis will be published elsewhere).

Figure 1. Rhabditid phylogeny as inferred by weighted parsimony jackknife analysis
using DNA sequences from three genes (SSU and LSU rRNA and RNA polymerase II). Strain designations are given for species available as stocks. Gray taxa names
designate outgroup representatives. Some of the clearer monophyletic groups are
delineated with brackets. Numbers on the branches indicate the percentage of 1,000
jackknife replicates in which the branch appeared (50% of randomly chosen sites were
deleted in each replicate). For each jackknife replicate, four random sequences of taxon
addition were used to generate starting trees. Branches were collapsed where jackknife
values were below 50%. The position of the strongylid clade, inferred from previous
analyses (Blaxter et al., 1998; Fitch and Thomas, 1997), is shown as
a dashed line. (KK and DF, unpublished; details of the analysis will be published
elsewhere.). Right-click or control-click to see larger image.

Two major clades of rhabditids can be distinguished, called here "Pleiorhabditis"
and "Eurhabditis" (Figure 1). Although these two groups were
combined in a single monophyletic group (supported by 87% bootstrap value) in an
earlier analysis using only SSU rRNA genes (Sudhaus and Fitch, 2001), such a clade
does not appear in the present jackknife analysis with additional genes.
Pleiorhabditis comprises several genera of species with a posterior vulva (e.g.,
Mesorhabditis, Teratorhabditis) and
also the genus Pelodera which includes Peloderastrongyloides, reported to be a facultative parasite of mammals
(Sudhaus and Schulte, 1988; Jones et al., 1991). Eurhabditis includes
almost all the other rhabditid species, including
Caenorhabditis, and thus differs from the Eurhabditis
originally proposed by Sudhaus on the basis of a different view of relationships
(see Sudhaus and Fitch, 2001). Additionally, Eurhabditis includes the "Protorhabditis
group", named after its most prominent members, which is the sister clade to
Caenorhabditis. Eurhabditis includes a monophyletic genus
Oscheius that comprises two separate clades, one of
leptoderan species, the Insectivora group, and another of
peloderan species, the Dolichura
group, as proposed by Sudhaus and Hooper (1994; see also Figure 4). One member of this latter group, Oscheius
tipulae (represented here by strain CEW1) is used as a satellite
model organism for comparative studies of development (see Evolution of development in nematodes related to C. elegans; Sommer, 2000) and molecular biology (e.g. Evans et al., 1997). The strongylid parasites of vertebrates and the
Heterorhabditis pathogens of insects have also clearly
evolved from within the Eurhabditis clade (see A quick tour of nematode diversity and the backbone of nematode phylogeny).

2.2. Relationships within Caenorhabditis

Relationships are now well-resolved among the Caenorhabditis
species which are represented by live strains (Figure 2; Kiontke et al., 2004; Cho et al., 2004). Of these species,
C. briggsae and C. remanei are closest
relatives. No single species is most closely related to C.
elegans, which is the sister species of a clade that includes
C. briggsae, C. remanei, and
Caenorhabditis n. sp. (represented here by strain CB5161).
The closest species to these four Elegans group species is
C. japonica, which was chosen for a genome sequencing
project in order to provide an outgroup comparison for the four
Elegans group species. Another undescribed new species
(represented here by strain PS1010), which has been used in some comparisons (e.g.
Baldwin et al., 1997), diverged earlier than C.
japonica. Uncertainty still exists for some
Caenorhabditis relationships; instead of being related in the
manner depicted in Figure 2, C. sp. PS1010 could be most closely
related to C. drosophilae and
Caenorhabditis n. sp. (DF5070) (Kiontke et al., 2004). For more about Caenorhabditis biodiversity and a
phylogenetic hypothesis incorporating other Caenorhabditis
species, see Ecology of Caenorhabditis species
(also Sudhaus and Kiontke, 1996).

Figure 2. Phylogeny of Caenorhabditis species represented by
living strains and four outgroup representatives. Black numbers on the branches denote percentage jackknife support from 2,000
replicates for an analysis incorporating all taxa shown using SSU and LSU rRNA genes and
RNAP2 with the third codon positions deleted. Red numbers denote jackknife support in
5,000 replicates for an analysis that included the seven most closely related
Caenorhabditis species for SSU and LSU rRNA genes, and coding regions from genes for
RNAP2, par-6, and pkc-3 with the third
codon positions included. Third codon positions were only saturated with substitutions
in pairwise comparisons involving species less closely related to C.
elegans than C. sp. PS1010. (Modified from Kiontke et al., 2004). Right-click or control-click to see larger image.

2.3. Strongylids and diplogastrids are rhabditids

Rhabditids, as a monophyletic group, include two taxa that have been previously
classified separately. First, the strongylids are a group of vertebrate parasites
traditionally considered outside of rhabditids. Molecular analyses have placed
strongylids clearly within the Eurhabditis group of rhabditids and as a sister group
of the insect pathogenic Heterorhabditis (Fitch and Thomas, 1997; Blaxter et al., 1998; Sudhaus and Fitch, 2001; De
Ley and Blaxter, 2002).

A second group of species traditionally classified outside of rhabditids is the
group designated here informally as diplogastrids (called Diplogastrina,
Diplogastridae, or Diplogasteromorpha in formal classification systems), which
includes the satellite model system, Pristionchus pacificus
(see Evolution of development in nematodes related to C. elegans). Specifically, there appears to be a relationship between diplogastrids
and Rhabditoides inermis, a rhabditid (see also Sudhaus and
Fitch, 2001). According to this phylogeny, R. inermis
is more closely related to diplogastrids than to its traditional congener, Rhabditoides inermiformis, despite their morphological similarities. (Another
species, Rhabditoides regina, resides within Pleiorhabditis,
thus making "Rhabditoides" polyphyletic.) Furthermore, our
phylogeny suggests that Poikilolaimus could be in the most
anciently diverged lineage of rhabditids, placing diplogastrids distinctly within
rhabditids, although the jackknife support (58%) is not strong for this conclusion.
Together, these data suggest that diplogastrids are not a clade separate from
rhabditids, but are instead derived from within rhabditids. These data contradict a
recent proposal based on morphology that bunonematids are the sister taxon of diplogastrids (Fürst von Lieven, 2002).

3. Examples of character evolution

3.1. Reproductive mode

Using the molecular phylogeny, we have traced the evolution of gonochorism (males
and females must cross to propagate), self-fertile hermaphroditism (see Sex-determination in the germ line), heterogony (alternating generations of gonochorism and hermaphroditism), and parthenogenesis (sperm, if they exist, do
not contribute genetic material; Figure 3). Among the taxa represented in Figure 3, we find that gonochorism is ancestral and that hermaphroditism has arisen at least ten times independently in all the
major rhabditid clades, with only one possible reversal back to gonochorism
occurring within the Dolichura species group of
Oscheius. Such massive convergence provides an excellent
opportunity to study how significantly developmental constraints limit or channel
evolution.

According to the most parsimonious reconstruction, and assuming reversibility and
equal chances of changes between character states, C. briggsae
and C. elegans evolved hermaphroditism independently (Kiontke et al., 2004). If we consider that changes from gonochorism to
hermaphroditism have occurred ten times more frequently than the reverse in
rhabditids, it becomes even more plausible that hermaphroditism evolved
independently in these two species. In such a case, there are two possibilities for
evolutionary change: parallelism (in which independent changes affect the same
mechanism) or convergence (in which the changes affect different mechanisms but
yield similar results). In the case of C. elegans and
C. briggsae, hermaphroditism appears to be convergent
because there is a fundamental difference between the two species in the identity of
FOG-2, for which no single ortholog exists in C. briggsae, and
developmental role of GLD-1, which inhibits female fate in the germline of
C. elegans but promotes female fate in C.
briggsae (Nayak et al., 2005; see Sex-determination in the germ line).

We also find that heterogony evolved twice (in
Heterorhabditis and an undescribed species represented by
strain SB347) and parthenogenesis evolved once (within the Protorhabditis
group).

3.2. Morphological evolution

As an example of morphological evolution, we have traced the history of changes in
morphogenesis of the male tail tip. In C. elegans, the shapes
of the hypodermal cells in the male tail tip change dramatically as the cells fuse
during L4 male morphogenesis and the conically pointed tail tip of the larva
retracts to form the rounded, "peloderan" tail of the adult
male (Nguyen et al., 1999). Tail morphogenesis does not occur in
hermaphrodites or in females of most other rhabditid species. Although males of most
rhabditid species do undergo tail morphogenesis, the tip of the tail does not always
retract. In such cases, the tail tip remains pointed, as in larvae, usually sticking
out beyond the posterior edge of the fan or bursa, if a fan exists. This tail form
is called "leptoderan".

Figure 3. Evolution of reproductive mode. Reproductive modes were determined for the terminal taxa and mapped onto the
molecular phylogeny depicted in Figure 1. Two Oscheius species and
the lineage to strongylids (dashed lines) were added according to their position in
phylogenies obtained with SSU rDNA alone. Red branches represent lineages in which
hermaphroditism evolved; blue branches represent lineages in which gonochorism occurred;
orange indicates heterogonic mode (different generations switch between gonochoristism
and hermaphroditism); green indicates probable or known parthenogens. Character states
for ancestral lineages were inferred by parsimony under the assumption that character
state changes are equally likely and reversible. Right-click or control-click to see larger image.

There have been at least seven independent changes in the shape of the male tail
tip during rhabditid evolution (Figure 4). Because some branches
are still unresolved, the directionality of most of these changes is unclear.
However, the present phylogeny allows us to conclude that at least two changes
occurred from peloderan to leptoderan. Some of the changes in
male tail tips must be convergent because we know that
peloderan tail tips can be constructed in different ways (e.g.,
with or without fusions of the hypodermal cells), as can leptoderan tail tips
(Nguyen et al., 1999; Fitch, 1997; Fitch, 2000). Because of
these convergences, it is clear that there is no monophyletic clade of leptoderan
species or of peloderan species (cf. Peloderinae of Andrássy, 1983). We have found that several other morphological characters (such as
lack of glottoid apparatus) show similar homoplasy at gross morphological levels
(e.g., Sudhaus and Fitch, 2001). Thus, rhabditids provide fertile ground for
investigating the role of developmental plasticity in evolution.

Figure 4. Evolution of male tail tip morphogenesis. Orange branches represent evolutionary lineages in which males had unretracted tail
tips, which are therefore pointed in shape and referred to as "leptoderan" if there is a
fan; green represents lineages in which males had retracted tail tips, which are thus
rounded in shape and are referred to as "peloderan," as in C. elegans. Gray represents
lineages for which the character state cannot be decided. Using the phylogeny depicted
in Figure 1, ancestral states were inferred by parsimony under the assumption that
character state changes are equally likely and reversible. Right-click or control-click to see larger image.

3.3. Intron evolution

Using the well-resolved Caenorhabditis phylogeny, we inferred
the most parsimonious history of intron gains and losses in a 1,860-bp portion of
the gene coding for the largest subunit of RNAP2 (Kiontke et al., 2004). This phylogeny of closely related species allows finer resolution of
such events than do comparisons among distantly related model organisms, which have
been the main staple of intron evolution fare (e.g. Rogozin et al., 2003). We find that intron evolution is extraordinarily dynamic (Figure 5). In this one region, the number of introns present in a
species can range between 1 and 14. Considering only homologous intron positions at
which a single most parsimonious reconstruction could be made ("unequivocal"
positions) and assuming that intron gains and losses are equally likely, introns
were lost 12 times and gained 4 times (Kiontke et al., 2004). If one
considers the possibility that losses are more likely than gains and that it might
be highly improbable for an intron to be regained in exactly the same spot, it is
possible that as many as 27 losses and at most 3 gains (but at least 1 gain)
occurred. Most of these intron losses occurred in the stem species of the
Caenorhabditis clade, and several losses occurred later as
the clade evolved (Figure 5). One intron in C. sp. CB5161 is unique
among all rhabditids we have characterized so far, and is thus a clear gain (Figure 5). Characterization of the homologous region in other
rhabditids suggests that intron evolution is very dynamic with periods of losses and
gains (KK and DF, unpub.). Frequent intron loss in other
Caenorhabditis genes has also been reported (Cho et al., 2004).
Recent intron gains have also been documented, some of which have sequence
similarity to other introns and could therefore have originated by reverse splicing
(Coghlan and Wolfe, 2004).

Figure 5. Intron evolution in part of the RNAP2 gene of
Caenorhabditis species. The presence (+) or absence (-) of
introns at particular sites (a-q) in the RNAP2 gene is depicted as a matrix (question
marks indicate missing data). For sites a, b, e, g, h, k-o, and q, a single,
unequivocal, most parsimonious history of intron insertion/deletion could be inferred
(rectangles on the branches of the phylogeny). For the other sites, more than one most
parsimonious scenario exists under the assumption that gains and losses are equally
probable. Sites at which a single deletion occurred during
Caenorhabditis cladogenesis are marked in dark blue; purple
indicates sites at which two deletions occurred; light blue and orange indicate sites at
which deletions and subsequent reinsertions could have occurred; red indicates a unique
intron insertion. (Modified from Kiontke et al., 2004). Right-click or control-click to see larger image.

3.4. Molecular divergence

Genome-level comparisons of molecular divergence show that C.
elegans and C. briggsae are a little bit less
similar at the molecular level than are human and mouse (Stein et al., 2003). We find the same relative differences when comparing these species
with RNAP2 and rRNA genes alone (Kiontke et al., 2004). Because many
taxa are represented by SSU rRNA sequences, we can use this molecule to compare the
degree of molecular divergences among other vertebrate taxa with the divergences
among rhabditids. The divergence between the least closely related
Caenorhabditis species (C. briggsae
versus C. sp. SB341) is comparable to the divergence between
mouse and zebrafish, encompassing a broad range of vertebrate lineages (Figure 6). Furthermore, species within the Eurhabditis clade are at
least as different at the molecular level as mouse and sea urchin, encompassing a
broad range of deuterostome lineages. It is important to note that the Eurhabditis
clade is but a small fraction of Nematoda. Thus, the nematodes diverged from other
animals very anciently, rates of molecular evolution are much faster in nematodes
than in deuterostomes, or both. Because there is no accurate method for calibrating
a "molecular clock" for nematodes, estimates for dates of divergence will be
extremely unreliable. Complicating this issue, there is considerable rate
heterogeneity among different lineages for some genes (e.g., SSU rRNA; see Kiontke et al., 2004). The best measure of taxonomic difference in rhabditids
is therefore simply the relative amount of sequence difference (as in Figure 6), not some date of divergence which could be very inaccurate.

Figure 6. (A) Comparisons of evolutionary divergences in SSU rRNA genes between
species pairs within nematodes (red) and deuterostomes (green). Pairwise divergences were estimated by summing lengths of intervening branches between
taxa in (B); standard deviations (error bars) were calculated as the square root of the
sum of the squares of the SDs of these branches. (B) Likelihood phylograms for SSU rRNA
genes used to calculate the divergences graphed in (A). Species relationships within
each group were assumed. A general time-reversible model (accounting for rate
heterogeneity across sites with some fraction of invariant sites, GTR+I+Γ) was used to estimate branch lengths (substitutions per site
± 1 standard deviation). Divergences
between any pair of taxa were then calculated as the sum of the branch lengths
separating those taxa on the tree. (Modified from Kiontke, 2004). Right-click or control-click to see larger image.

4. Resources for comparative biology using rhabditids

Arguably the most important tools for comparative studies using emerging model systems
are the organisms themselves. Genome sequences, interactome maps, cDNA libraries, and
other such molecular data and resources are important tools for functional genomics.
Ultimately, however, in vivo understanding of gene and genome
function - particularly with respect to mechanisms and patterns of gene expression,
development and behavior - requires analysis in the context of the living system itself.
Powerful comparative approaches depend on the availability of multiple, phylogenetically
well-defined, living species with clearly defined, standardized strains that can be
archived and shared. The Caenorhabditis Genetics Center (CGC) provides many stocks of wild strains that can be easily maintained cryogenically. We are
also developing a stock collection of rhabditid species, the New York University Rhabditidae Collection (NYURC) that is as
thoroughly representative of rhabditid phylogeny as possible. The NYURC curates strains
that are difficult to culture or archive cryogenically along with data about ecology,
biology, morphology, etc., for all rhabditids in culture.

5. Acknowledgments

We thank the National Science Foundation and the Human Frontier Science Program for
supporting this work. Although many people have been important to this work, we
especially thank Marie-Anne Félix, W. Kelley Thomas and Walter Sudhaus for their important collaborations and
contributions.
We thank Byron Adams and Steve Nadler for providing excellent suggestions in their
reviews of the manuscript.

*Edited by Lisa R. Girard. Last revised July 27, 2005. Published August 11, 2005. This chapter should be cited as: Kiontke,
K. and Fitch, D.H.A. The Phylogenetic relationships of Caenorhabditis and other rhabditids (August 11, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.11.1, http://www.wormbook.org.

†Terms used: taxon (plural taxa)- a group of phylogenetically related species; monophyletic- a group of species that includes
all descendants of one ancestor; paraphyletic- a group of species that includes some but not all descendants of an ancestor;
polyphyletic- a group of species that includes descendants of more than one ancestor; clade- a monophyletic taxon.